International Journal of Molecular Sciences Review Molecular Regulatory Mechanism of Exocytosis in the Salivary Glands Akiko Suzuki 1,2 and Junichi Iwata 1,2,3,* 1 Department of Diagnostic & Biomedical Sciences, The University of Texas Health Science Center at Houston School of Dentistry, Houston, TX 77054, USA; [email protected] 2 Center for Craniofacial Research, The University of Texas Health Science Center at Houston School of Dentistry, Houston, TX 77054, USA 3 Program of Biochemistry and Cell Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA * Correspondence: [email protected]; Tel.: +1-713-486-2641 Received: 1 September 2018; Accepted: 11 October 2018; Published: 17 October 2018 Abstract: Every day, salivary glands produce about 0.5 to 1.5 L of saliva, which contains salivary proteins that are essential for oral health. The contents of saliva, 0.3% proteins (1.5 to 4.5 g) in fluid, help prevent oral infections, provide lubrication, aid digestion, and maintain oral health. Acinar cells in the lobular salivary glands secrete prepackaged secretory granules that contain salivary components such as amylase, mucins, and immunoglobulins. Despite the important physiological functions of salivary proteins, we know very little about the regulatory mechanisms of their secretion via exocytosis, which is a process essential for the secretion of functional proteins, not only in salivary glands, but also in other secretory organs, including lacrimal and mammary glands, the pancreas, and prostate. In this review, we discuss recent findings that elucidate exocytosis by exocrine glands, especially focusing on the salivary glands, in physiological and pathological conditions. Keywords: exocytosis; salivary glands; Sjögren’s syndrome; protein secretion; membrane trafficking 1. Introduction The major (submandibular, sublingual, and parotid) and minor salivary glands produce 0.5–1.5 L of saliva daily, which is essential for oral health. Saliva comprises 99.5% of water, 0.3% of proteins, and 0.2% of both inorganic and organic substances [1]. The solid components differ from person to person, and from time to time in the same person. For example, salivary glands secrete 1.5 to 4.5 g proteins daily at a concentration of 0.47 ± 0.19 to 2.67 ± 0.54 mg/mL [2]. The protein concentration in saliva is lower than that in tears, which is around 8 mg/mL under basal conditions [3,4]. Acinar cells are a major cell type within the salivary glands, responsible for the production and secretion of prepackaged secretory granules that contain key functional salivary components, such as amylase, mucins, and immunoglobulins [5]. These salivary components are functionally important for the digestion and taste of foods, lubrication, buffering, and prevention of dental caries, periodontitis, candidiasis and halitosis (bad breath). This secretion process in exocrine glands, called exocytosis, involves secretory vesicle trafficking, docking, priming, and membrane fusion [6]. A failure during any of the steps in exocytosis in the salivary glands results in altered secretion of salivary proteins (Table1). Interestingly, several studies show that secretion of salivary proteins is reduced, and the contents of saliva are also changed, in patients with dry mouth syndromes (e.g., Sjögren’s syndrome) and in a mouse model for Sjögren’s syndrome (e.g., non-obese diabetic (NOD) mice) [1,7–10]. This evidence suggests that exocytosis may Int. J. Mol. Sci. 2018, 19, 3208; doi:10.3390/ijms19103208 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 3208 2 of 16 be altered in various conditions, and the altered salivary contents may constitute a risk factor for major oral health issues [11,12]. There are two types of acinar cells—serous and mucous acinar—in the salivary glands. Serous acini are composed of typically 8–12 pyramid-shaped cells containing many secretory granules in the apical cytoplasm, a well-developed rough-endoplasmic reticulum (rough-ER), and pronounced round nuclei in the middle of the cytoplasm. They secrete serous saliva, which contains digestive α-amylase (AMY1A), a protein that is crucial for food digestion [13,14]. By contrast, mucous acinar cells are larger than serous acinar cells and have flat nuclei towards the basal cell surface due to large numbers of mucin granules aggregated in the apical cytoplasm [13,14]. Mucous saliva contains mucins that are important for lubrication and oral health [13,14]. The salivary glands secrete saliva rich in salivary proteins, the diverse functions of which maintain oral health by providing lubrication, initiating digestion, and offering first-line immunity. Therefore, disruption of salivary gland functions quickly results in widespread deterioration of oral health. Despite the important physiological functions of salivary proteins, we know very little about the regulatory mechanism(s) of exocytosis in the salivary glands. A detailed understanding of the mechanism(s) by which exocytosis is regulated will provide new knowledge of its key function(s), not only in the salivary glands but also in other secretory organs, including the lacrimal and mammary glands, the pancreas, and prostate, in physiological and pathological conditions. Ultimately, this approach will identify novel targets for therapeutics and contribute to new diagnostic tools for identifying exocytosis defects in at-risk populations such as those with high cholesterol levels or who have high-cholesterol diets. Sjögren’s syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the exocrine glands, mainly the salivary and lacrimal glands, which results in reduced secretory functions and oral and ocular dryness [15,16]. The diagnosis is based on a combination of symptoms, physical examination, and blood tests (American-European Consensus Group, AECG, 2002; Sjögren’s International Collaborative Clinical Alliance, SICCA, 2012). While the pathogenesis of Sjögren’s syndrome remains elusive, various factors, including environmental, genetic and hormonal factors, seem to be involved, and either immune cells or exocrine gland cells are primarily damaged or disorganized to induce inflammation in the salivary and lacrimal glands [17–19]. Table 1. Phenotype in mice with deficiencies in genes related to the exocytosis process. Mutant Mouse Phenotype Gene Name Reference Salivary Acinar Cells Lacrimal Acinar Cells Pancreatic Acinar Cells Accumulation of secretory Accumulation of Accumulation of secretory Vamp8 [20] vesicles secretory vesicles vesicles Syntaxin2 Not studied Not studied Increased exocytosis [21] Enlarged secretory Rab3d Enlarged secretory vesicles vesicles Enlarged secretory vesicles [22,23] Decreased total protein amount in tears Increased total protein Rab27a Not studied Not studied [23,24] amount in tears Reduced number of Rab27b Not studied secretory vesicles Not studied [23,24] Increased total protein amount in tears Accumulation of enlarged Accumulation of enlarged Noc2 Not studied [25,26] secretory vesicles secretory vesicles Gland degeneration Gland degeneration Sec23b Not studied [27–29] Absent or reduced number Absent or reduced number of secretory vesicles of secretory vesicles Int. J. Mol. Sci. 2018, 19, x 3 of 16 Int. J. Mol. Sci. 2018, 19, 3208 3 of 16 2. The Exocytosis Process 2. TheExocytosis Exocytosis is Processa dynamic secretion process consisting of vesicle trafficking, tethering, docking, priming,Exocytosis and fusion is a [6,30]. dynamic After secretion proteins process are folded consisting in the ER, of they vesicle leave trafficking, this organelle tethering, inside docking, vesicles priming,coated with and the fusion coat [protein6,30]. Aftercomplex proteins II (COP areII) folded towards in the ER,Golgi they apparatus. leave this Through organelle the insidetrans- vesiclesGolgi network, coated withproteins the coatare proteintransported complex to variou II (COPII)s destinations towards theby Golgia variety apparatus. of mechanisms. Through For the transexample,-Golgi the network, destination proteins of the arediffe transportedrent proteins to is various determined destinations by the molecule by a variety coats ofon mechanisms. a vesicle, by Foractin-dependent example, the motors destination (Myosins), of the and different by microt proteinsubule-dependent is determined motors by (Kinesins the molecule and Dyneins) coats on a[31,32]. vesicle, During by actin-dependent intracellular transportation, motors (Myosins), RAB and GTPases by microtubule-dependent and their effectors regulate motors (Kinesinsthe secretory and Dyneins)vesicle movement [31,32]. During throughout intracellular the cytoskeleton transportation, [33]. RAB GTPases and their effectors regulate the secretorySoluble vesicle N-ethylmaleimide-sensitive movement throughout the factor cytoskeleton attachment [33]. protein (SNAP) receptor (SNARE) proteins,Soluble suchN -ethylmaleimide-sensitiveas vesicle-associated membrane factor attachmentproteins (VAMPs), protein (SNAP)are present receptor in the (SNARE) secretory proteins, vesicle suchmembrane as vesicle-associated (Figure 1). The membrane secretory vesicles proteins are (VAMPs), transported are present from the in the trans secretory-Golgi network vesicle membrane via actin- (Figureand microtubule-based1). The secretory molecular vesicles motor are transported proteins towards from the plasmatrans-Golgi membrane. network Once via the actin- secretory and microtubule-basedvesicles arrive to the molecular plasma motormembrane,